Seasonal influenza epidemics result in significant morbidity and mortality and impose a large societal burden associated with medical care and work loss. Approximately one third of influenza cases are due to influenza B virus (IBV), and IBV is associated with more severe disease in children and higher rates of hospitalization than seasonal A/H1N1 viruses. As in influenza A virus (IAV), the constant evolution and antigenic drift of IBV necessitates annual updates in the two B lineage components of the quadrivalent vaccine. Phylogenetic studies of viral sequences collected through global surveillance indicate that IBV has a slower rate of antigenic evolution than IAV, yet the reason for these differences are unclear. Ultimately, the generation and early transmission of novel variants is dependent on processes that take place on the scale of individual hosts. Very little is known about these host-level dynamics in IBV. The long-term goal of this research is to elucidate the molecular evolution of influenza viruses within hosts and to understand how novel variants spread between them. The objective of this exploratory project is to determine the extent to which the differences in evolutionary rates between IAV and IBV are explained by differences in the mutation rate, which determines how rapidly new variants arise, and the transmission bottleneck, which determines how quickly a new variant will move through host populations. The feasibility of the proposed research is supported by published preliminary data, which demonstrate (i) the development of a novel and precise mutation rate assay that provides unbiased estimates of each nucleotide substitution class, (ii) the assay's specificity and power for distinguishing the rates of mutation for each nucleotide substitution class in closely related viruses, (iii) the use of high quality next generation sequence (NGS) data of viruses from naturally infected individuals to identify transmission pairs and to quantify the transmission bottleneck. Detailed analyses of host level IBV evolution will be accomplished in two aims. (Aim 1) Determine the rates for all mutational classes in both B/Yamagata and B/Victoria viruses. A novel and precise mutation rate assay will be used to define the rates for all 12 mutational classes in both IBV lineages. (Aim 2) Define the molecular genetics of transmission for influenza B viruses in a community cohort. NGS of viruses from index cases and household contacts will be used to define household transmission pairs and to quantify the size of the transmission bottleneck. This research is innovative, because it will apply a newly developed assay to determine the mutation rates of IBV in unprecedented detail and will leverage a unique household cohort to define the virus' transmission dynamics in natural infection. The proposed research is significant, because it will define the evolutionary dynamics of IBV at the level of the individual host and enable better predictive models for seasonal vaccine design. This will bring us closer to a universal influenza vaccine.